The overall goal of this proposal is to examine the different Ca 2+ signaling mechanisms that have evolved in atrial cells. Atrial and ventricular myocytes differ with respect to speed of contraction, development of t-tubules, IP3Rs, ANP secretion, and automaticity. In atrium the DHPRs and RyRs are co-expressed only at the sarcolemma, leaving the centrally located RyRs unassociated with Ca 2+ channels and possibly less directly controlled by ICa. Though IP3Rs are expressed 6-fold higher in the atrium vs. ventricles, their spatial or functional contribution to atrial contractility remains unclear. Further, the role of stretch-activated atrial channels or NO/NOS signaling reported to modulate spontaneous Ca 2+ spark frequency and contractility, and their interaction with the Ica-gated Ca 2+ signaling, remains untested. Based on preliminary results we propose to study "multimodal activation" of Ca 2+ signaling in atrial cells and challenge the conventional view of Icagated release of Ca 2+ at cell periphery initiating the regenerative wave of CICR into the cell center. We hypothesize that atrial focal Ca 2+ release sites are differentially controlled in junctional and non-junctional SR, that these mechanisms are related to atrial cell types, variability in activation of Ca 2+ pools, the level of expression of IP3Rs, the modality of gating and relevant to signaling of myocyte hypertrophy. Using confocal and TIRF Ca 2+ imaging in voltage-clamped atrial cells in Aim 1 of the proposal we shall examine: 1) direct vs. indirect gating by Ica of peripheral and central Ca 2+ stores, 2) characterize Ca 2+ release by IP3, and 3) determine role of rudimentary t tubules through structure-function studies in rat, cat and guinea pig atrial cells. In Aim 2, peripheral vs. central Ca 2+ sparks will be compared with respect to: a) abrupt vs. gradual termination of Ca 2+ release, b) reactivation or restitution, c) role of CICR, and IP3. In addition, peptide fragments of the carboxyl tail of alC with specific Ca 2+- and calmodulin-binding motifs will be dialyzed into the cell in order to study possible steric interactions between DHP and RyRs, particularly in the central release sites. In Aim 3, we will characterize the signaling cascade responsible for novel Ca 2+ transients activated by mechanical shear of rapid solution flow, identify the responsible Ca 2+ stores, quantify the unitary properties of its Ca 2+ sparks, map their spatial distribution, gating, and association with central and peripheral release sites and atrial cell types, and determine their relation to Ica-gated Ca 2+ pools and atrial contractility.